PROJECT SUMMARY Discovering the genetic basis of drug response is creating new opportunities to repurpose old drugs for the treatment of different cancers. We have discovered DNA repair pathways that determine the sensitivity of mammalian cells to a thiopurine drug named 6-thioguanine. Thiopurines have been used for over half a century to successfully treat pediatric cancers but otherwise are not used to treat other cancers. The goal of this project is to delineate the genetic mechanisms responsible for the cellular response to thiopurine treatment. This information will then lead to novel and rational therapeutic combinations based on knowledge of defective genetic pathways in tumor cells that are targetable with a ?repurposed? drug. Homologous recombination (HR)-mediated repair suppresses chromosomal instability and cell death induced by many DNA damaging agents, including thiopurines. We showed that cells knocked out for an important HR protein, RAD51D, were extremely sensitive to the toxic and chromosome-damaging effects of 6-thioguanine. RAD51D is part of the RAD51 family of genes broadly appreciated to be indispensable for HR, yet the protein products of the paralogs have poorly understood, specialized functions. Human cancer susceptibility alleles for breast and ovarian cancer have been identified in RAD51B, RAD51C, and RAD51D, which strongly suggest that these mutations create an Achilles? heel that is potentially exploitable with thiopurine treatments. The hypothesis to be tested is that RAD51 pathway genes promote HR-mediated repair efficiency, and thereby dictate the extent of thiopurine-induced chromosomal damage and cellular survival. The hypothesis will be tested in Aim 1 by determining which of the five RAD51 paralog genes contribute to suppressing 6- thioguanine induced chromosomal instability and promoting RAD51-dependent HR. An additional benefit to generating paralog knockouts in the same background is to identify paralog-specific HR subpathways. This will be achieved by measuring cellular sensitivity and HR in response to other DNA damaging agents: ionizing radiation, which causes double strand breaks, and cisplatin, which causes inter-strand crosslinks. Aim 2 will test selected, clinically relevant, human variant alleles in these genes to determine whether these alleles are ?druggable,? i.e., cause chemo-sensitivity. An additional benefit is to look for novel separation of function mutations in the variant alleles. During this project, undergraduate, graduate, and Doctor of Pharmacy students will receive outstanding research training in cancer pharmacology and pharmacogenetics. Undergraduate and Doctor of Pharmacy students are recruited from the University of South Carolina Honors College, which is ranked first by the Public University ?Review of Fifty Public University Honors Programs.? The long-term benefit to human health will be the identification of genetic profiles that confer susceptibility to thiopurine drugs, which can then guide clinical decisions to create individualized thiopurine treatment strategies.